Een eenvoudige Behavioral Assay for Testing visuele functie in Xenopus laevis

The overall goal of this procedure is to test the visual function of tadpoles by recording the amount of time the animal spends on half of the testing tank that is covered in black. This is accomplished by first raising tadpoles to stages 45 to 50. The second step is to set up a half black, half white tank along with a camera for the behavior assay.

Next place a second tank with a tadpole into the outer tank. For each tadpole record, the vision guided behavior as the background in its environment is changed. The final step is to perform a retinal ex sodomy on the tadpoles and then repeat the behavior assay.

Ultimately, data from this simple, inexpensive assay are used to show the tadpoles have a preference to swim on one side of the tank over the other, under tests of phototop vision. The main advantage of using this method over other techniques like the optic kinetic response or the tail flip assay, is that it is an inexpensive, simple method that uses equipment readily available in most opus laboratories. To prepare the tadpoles, first, obtain fertilized xenopus embryos, put mark's, modified ringers, or MMR diluted by a factor of 10 in 60 millimeter Petri dishes with 50 micrograms per milliliter.

Gentamycin antibiotic place blastula to neural plate stage embryos. Stages two to 15 at 18 degrees Celsius in the Petri dishes. Once embryos reach a new cup in favor stage of about 27 to 30, switch them to 0.1 XMMR without antibiotics to allow gut bacterial growth.

When the tadpoles reach the feeding stage around stage 45, move them into 100 millimeter Petri dishes with 0.1 XMMR. The next day, feed them nettle powder supernatant. After one week in the 100 millimeter Petri dish, fill a half gallon tank with frog water.

Move animals to the tank and continue the nettle powder supernatant feeding schedule, but change the water only two to three times per week. When the animals are in half gallon tanks, maintain them in a 12 hour light, 12 hour dark cycle with lights turning on at 6:00 AM Prior to testing, place the half gallon tank with the test subjects on a white surface overnight for at least 12 hours prior to testing. The behavior assay should be done in a quiet, low traffic area with standard fluorescent lighting.

Prepare two nesting half gallon testing tanks on the outside corners of what will be the inner tank. Use a Sharpie to mark the waterline at five centimeters above the bottom. Also for the inner tank, identify any divots in its interior.

Fill these with an inert compound to prevent tadpoles from lingering in them. Now turn attention to the second outer tank. For the next step, obtain black electrical tape and white tissue paper.

Use the tape to cover the exterior of one half of the tank and the tissue paper to cover the exterior of the other as shown here. Once the tanks are prepared, set up an inoculating turntable and place the nested tanks on it. Position a tripod and webcam so that the camera can image the interior of the tanks from above.

Connect the webcam to a computer with video capture and playback software. Turn on the camera and prepare to make a new movie recording. Next drape a lightweight cotton cloth over the camera and tanks.

To reduce external cues to the tadpoles, use luminance meter and check that the luminance under the cloth is between 35 to 50 candles per square meter. Start the behavior assay by retrieving the inner testing tank. Fill it with frog water to the five centimeter mark.

Use a net to transfer an animal from its half gallon container to the inner testing tank at the computer. Make certain the recording software is open and recording the testing area for record keeping. Write the animal name, the date, and the time on a piece of paper.

Place this under the camera for recording. After this is done, preset a timer for two minutes. Next, orient the outer tank on the turntable, so the black side is on the right.

Now place the test tank into the outer tank. In the recording area, close the drape and use the computer screen to ensure the orientation of the outer tank. Make sure video is being recorded and start the timer.

When the timer beeps, keep the drape in position and remove the inner test tank. Next, rotate the outer tank 180 degrees so that the black side is now on the left. Return the test tank to the outer tank and start the two minute timer.

Again, remove the inner tank. Rotate the outer tank by 180 degrees, and replace the inner tank every two minutes for a total of 10 times. When done, stop the video recording.

Return the tadpole to its half gallon tank. Prepare a microscope for performing the retinol ex sodomy. Fill a 60 millimeter Petri dish with 1%aros melted in 0.1 XMMR and let it solidify.

Remove a small rectangular piece of the aros the size of the tadpole. When ready to proceed, retrieve the test animal and place it in 0.02%Trica. Continue when it is unresponsive to a tail pinch with number three forceps.

Then transfer the animal to the dish. Proceed by mounting the Petri dish on the microscope. Prepare to brace the animal with forceps on one side.

On the opposite side, use a 25 gauge needle to pierce the skin behind the dorsal region of the eye at a 45 degree angle, carefully reach into the hole with number five, forceps and snip the optic nerve while avoiding the vein next to it. Flip the nerve out of the way and retract the forceps after surgery. Recover the animal by placing it in a 100 millimeter Petri dish with 0.7 XMMR and 50 micrograms per milliliter.

Gentamycin for 20 minutes. After that, transfer the tadpole to a recovery tank with frog water. Place the tank on a white surface in preparation for testing.

Let the animal rest overnight. The next morning use the behavioral assay to test visual function. This movie shows representative data for the experiment.

On the left are the animals before surgery on the right are the same animals after retinal axons from both eyes have been severed. This is indicated by the schematic in the bottom corners. The water was disturbed to show that nothing has been added to repel the animals from the black side or attract them to the white side.

The pre-surgery tadpoles respond to the background color in less than 30 seconds while the blind tadpoles swim aimlessly around the tank. This chart shows the percentage of time spent in the white and black regions for a single animal under three conditions. First, the animal was observed before any surgery using the assay.

On two consecutive days after the right optic nerve was severed, its behavior was observed over two days of testing. Finally, after both optic nerves were severed, there were two more days of observation with the assay. The animal spent more days on the white side of the tank when at least one optic nerve was intact.

After watching this video, you should have a good understanding of how to test the vision of tadpoles using simple reagents and equipment found in most frog labs. Good luck with your experiments.